Since the development of recombinant DNA techniques in the late 1970s, gene fusion technology has been used for generating multifunctional proteins for a broad range of applications. Fusion proteins are utilised in protein science research for diverse applications. In order to construct such fusion proteins, two types of connection are possible. One is ‘end to end’ fusion in which the N-terminus of one domain is linked to the C-terminus of the other domain. The second is “insertional” fusion in which one domain is inserted in-frame into the middle of the other parent domain.
Antibodies have widely been utilized as reagents for the research for the purpose of detection, purification, elimination, inhibition of a protein or the like, because it has property of recognizing specific protein and binding thereto. Recently, it has widely been used not only as reagents for the research but also as drugs or diagnostic agents.
In producing antibodies, it has so far been general to use a method that a large amount of protein as an antigen is purified and injected to an animal or animals such as rabbits or mice to collect antibodies generated in sera. It required, however, much time and a great deal of labor to obtain a large amount of a purified antigenic protein. It is desired to provide a more convenient method for producing antibodies, accordingly.
It was reported that when a gene coding for an influenza virus nucleoprotein is integrated into an expression vector and intramuscularly injected directly as DNA to mice, then virus proteins are produced in the murine bodies and additionally the antibody against these proteins are generated in the sera (Ulmer et al., Science 259: 1745-1749, 1993). As a result, this expression vector received much attention for the use to produce antibodies. Thus, it has been designated as gene immunization that an expression vector for an antigenic protein is inoculated directly to an animal to generate and prepare antibodies. In using gene immunization, however, in some cases, the titer of the generated antibody is very low or no antibody is generated depending on the kind of the antigen used.
It was reported as an example of gene immunization that ovalbumin was fused in the downstream of transmembrane domain of transferrin receptor to form a membrane type and it was injected intramuscularly or subcutaneously to mice in order to investigate an effect of the expression site of antigenic protein on the efficacy of gene immunization. The titer of the antibodies generated, however, rather decreased since the protein was converted into a membrane type. (Boyle et al., Int. Immunol. 9: 1897-1906, 1997).
The document EP 1 304 375 refers to a method for producing an antibody against an antigenic protein, wherein the antigenic protein is fused with the C-terminal side of a transmembrane domain of which the N-terminal side is located in the cell and the C-terminal side is out of the cell and the fusion protein is inoculated in a non-human animal.
The purpose of the invention of the present application is to provide an improved method for producing antibodies directed to proteins, for which it was difficult to produce antibodies in so far known gene immunization methods.
Additionally, the purpose of the invention is to provide an expression vector useful to express an immunogen which is applied in the above-mentioned method for producing an antibody.
How these and other aspects of the invention are achieved will be seen from the disclosure which follows.